Despite a number of carefully designed studies, the fundamental question of the role of amino acids and insulin in the regulation of protein synthesis in human muscle still remains unclear. Although insulin has been shown to stimulate muscle protein synthesis in growing rats, similar experiments in humans have failed to show any change, leading to a proposal that in humans a supply amino acids is also needed for insulin to stimulate protein synthesis. Experiments to prove this theory by infusing an amino acid mixture together with insulin have given conflicting results with the two different labeled amino acids used to assess protein synthesis. The proposed project aims to resolve this discrepancy by investigating an alternative hypothesis, which attributes the conflicting data to an artifact of the technique for measuring protein synthesis, resulting from changes in the concentrations of the tracer amino acids. This could lead to changes in the isotopic enrichment of the precursor pool, and hence to artifactual changes in the apparent rate of protein synthesis. The hypothesis will be tested by infusing three different labeled amino acids and characterizing the relationship between the apparent response of muscle protein synthesis to insulin infusion and the plasma concentrations of the individual tracer amino acids used for measurement. As real changes in protein synthesis should be detected equally by any tracer, rates will be assessed by giving three different labeled amino acids. Changes in concentration of each tracer will be manipulated by infusing single amino acids, enabling real changes in protein synthesis to be distinguished from changes in precursor enrichment. Rates of protein synthesis will also be measured by a technique which simplifies the assessment of precursor enrichment, the flooding technique. In addition changes in enrichment of the precursor for protein synthesis with changing tracer concentration will also be investigated directly in experiments in dogs where aminoacyl-tRNA measurements are possible. The results of these studies will amplify our understanding of the mechanism of insulin action by indicating whether protein synthesis in human muscle is sensitive to stimulation by insulin, and whether a continuous supply of amino acids is required. This knowledge will be important in understanding clinical conditions where muscle protein is lost, such as diabetes, trauma, and cancer.